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HCN 1
HCN 1
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been
linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been
reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate
HCN1-related disorders and investigate genotype–phenotype correlations further, we assembled a cohort of 33 unpublished pa-
tients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families
with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals.
Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness.
Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies
and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise un-
classified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: one adopted
patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense
variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with
severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes
were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants
were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged
from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence. Functional analysis of three
Received March 22, 2018. Revised July 13, 2018. Accepted August 9, 2018
ß The Author(s) (2018). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For permissions, please email: journals.permissions@oup.com
HCN1 mutation spectrum BRAIN 2018: 141; 3160–3178 | 3161
different substitutions altering Gly391 revealed that these variants had different consequences on channel biophysical properties.
The Gly391Asp variant, associated with the most severe, neonatal phenotype, also had the most severe impact on channel function.
Molecular dynamics simulation on channel structure showed that homotetramers were not conducting ions because the permeation
path was blocked by cation(s) strongly complexed to the Asp residue, whereas heterotetramers showed an instantaneous current
component possibly linked to deformation of the channel pore. In conclusion, our results considerably expand the clinical spectrum
related to HCN1 variants to include common generalized epilepsy phenotypes and further illustrate how HCN1 has a pivotal
function in brain development and control of neuronal excitability.
38 Department of Pediatric Neurology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, ULB, Brussels,
Belgium
39 Department of Neuroscience, Columbia University, New York, NY 10032, USA
40 Membrane Biophysics, Deparment of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany
41 Department of Neurology, San Gerardo Hospital, University Milano-Bicocca, Monza, Italy
42 IGBMC, CNRS UMR 7104/INSERM U964/Université de Strasbourg, 67400 Illkirch, France
43 Institute of Human Genetics, University Hospital Essen, University Duisburg-Essen, Essen, Germany
Patients and families were recruited from epilepsy and genetic experiments were performed at room temperature 24 h after
centres around Europe (Italy, France, Netherlands, Germany, transfection, as previously described (Nava et al., 2014a). For
UK, Czech Republic, Portugal, Switzerland and Belgium), co-expression experiments, an equal amount of wild-type and
Brazil, USA, Canada and Australia. Patients 11 and 36 were mutant constructs (0.5 mg of each plasmid) was transfected in
recruited though Decipher (ID: 260229 and 357848) (Firth the same conditions.
et al., 2009). Collection and analysis of retrospective clinical, The biophysical properties of M305L, G391C, G391D,
EEG, neuropsychological and neuroimaging data were assessed G391S and I397L were further studied in HEK293T cells tran-
using a specific format filled by the treating specialist aiming to siently transfected with wild-type and/or mutant constructs
obtain accurate and homogenous information. We classified seiz- using TurboFectTM transfection reagent (Thermo Fisher).
ure types and epilepsy/syndromes according the International Either 1 mg or 0.5 mg of the HCN1-containing vector
Van der Waals forces were switched to zero between 0.8 and 1.2 have had prolonged febrile seizures and hemiclonic compo-
nm. Electrostatic interactions were represented using the fast nent in one. Six patients (31.5%; Patients 1, 3, 10, 11, 16
smooth particle-mesh Ewald (PME) method with a Coulomb and 19) had generalized seizures including absences, eyelid
cut-off of 1.2 nm (Essman, 1995). Hydrogen bonds were kept
myoclonia and afebrile tonic-clonic seizures. In five probands
constant using the LINCS algorithm (Hess, 1997). The velocity-
(26.3%; Patients 8, 9, 12, 13 and 15), initial seizures were
rescale thermostat (Bussi et al., 2007) with a coupling time con-
stant of 0.1 ps was used to keep the simulation temperature at classified as possibly focal with a variable combination of
298 K. The pressure was kept at 1 bar using the Parrinello- symptoms including hypotonia, hypomotor behaviour,
Rahman barostat algorithm (Parrinello, 1981) with a coupling apnoea and cyanosis, tonic posturing, clonic jerking with or
constant of 5 ps. Prior to 100 ns production simulation, the without secondary generalization. In Patient 17, characteriza-
Figure 1 Schematic representation of HCN1 variants on the gene and protein. (A) Location of variants identified in this study (above)
or previously reported (Nava et al., 2014; below) on schematic representations of the HCN1 coding exons (NM_021072.3) and corresponding
protein domains. Variants in black correspond to de novo variants identified in EIEE or GGE patients. Variants in blue are dominantly inherited
within GGE-GEFS+ families. Variants in orange are of unknown significance. (B) Location of HCN1 variants on a schematic representation of the
HCN1 channel. Variants indicated with a star are present in an adopted individual with severe EIEE. (C) Comparison of the variants identified in
patients with neurodevelopmental phenotypes (above) and variants present in ExAC (below), showing that in control populations, variants
clustered in N- and C-terminal regions of the HCN1 protein, which are more tolerant to genetic variation while pathogenic variants tend to
cluster to functional or transmembrane domains of the channel.
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Table 1 Clinical and genetic summary of the 19 sporadic patients with de novo HCN1 variants
Patient Gender Age at Seizure Seizure type Seizure type at follow-up Sensitivity Seizure Resistance Development Epilepsy type/ Mutation
number the study onset at onset to fever frequency to AEDs syndromes
1 F 12 y 6 m 12 m TCS TCS, At No Daily Yes Severe ID EIEE p.Phe143Tyr
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At = atonic; CAE = childhood absence epilepsy; Cl = clonic; DD = developmental delay; F = female; Fc = focal; FS = febrile seizures; FS+ febrile seizure plus; Gen = generalized; ID = intellectual disability; LOC = loss of consciousness; M = male;
MA = myoclonic-atonic; My = myoclonic; NA = unavailable; NOEE = neonatal-onset epileptic encephalopathy; Tn = tonic; TCS = tonic-clonic seizure(s); Uncl = unclassified.
Detailed clinical information, including Brain MRI, EEG findings and antiepileptic treatment is provided for Patients 1–19 in Supplementary Table 2.
C. Marini et al.
Family/ Gender Age at Seizure Seizure type Seizure at follow-up AEDs EEG Brain MRI Development Epilepsy type/ Mutation
patient study onsset syndromes
number
Family T
20 M 66 y 8 m FS and afebrile TCS Seizure until age 6 y then single PB, currently no AEDs Normal NA NA GEFS+ p.Thr171Arg
TCS in adult age
21 M 32 y 8 m FS; TCS, possible Seizure-free from age 12 y PB + GVG + VPA Normal Normal Borderline GEFS+
focal onset 4TCS currently no AEDs
22 F 21 y 10 m FS FS and afebrile TCS, seizure-free VPA, ESM, currently GSW Normal Mild ID (TIQ = 50) GEFS+
for 43 y no AEDs
23 F 21 y 11 m FS FS and afebrile TCS, seizure-free VPA Normal Normal Mild ID (TIQ = 66) GEFS+
for from 10 to 18 y
Family M1
24 F 49 y 3 m FS, febrile, afebrile Seizure-free from age 12 y after CBZ NA NA Normal GEFS+ p.Cys329Ser
TCS CBZ
25 F 29 y 1 y FS Seizure-free after VPA VPA NA NA Normal FS
26 F 6 y 18 m TCS without fever Status epilepticus episode treated VPA Normal Normal Normal GGE
by PHT then seizure-free on VPA
27 M 23 y 8 m Febrile TCS FS and afebrile seizure and absence VPA Normal Normal Mild ID (TIQ = 52; GGE
from age 4 y; non-progressive VIQ = 46; PIQ = 70)
action myoclonus from age 8 y;
seizure-free after VPA
28 F 20 y 13 m FS Seizure-free with CBZ until 13 y; CBZ Normal Normal CT scan Normal GEFS+
CBZ was suspended: seizure
recurrence. Seizure-free after
CBZ reintroduction
Family M2
29 F 8 y 18 m FS, febrile and Seizure-free after VPA VPA Fast activity Normal Borderline (WIPPSI: GEFS+ p.Val414Met
afebrile TCS IQ = 77)
30 F 5 y 18 m FS Seizure-free No AEDs Rare focal PA, Normal Borderline (DQ GEFS+
fast activity Griffith scale = 70)
31 M 59 y 12 m FS Seizure-free No AEDs NA NA Normal FS
Family O
32 F 7 y 7 y TCS Seizure-free VPA GSW during IPS Normal Normal GGE p.Ser680Tyr
33 F Unknown Childhood Absence Absence and single TCS in No AEDs GSW Normal Normal CAE
childhood, then
seizure-free
AED = anti-epileptic drug; CBZ = carbamazepine; DQ = developmental quotient; ESM = ethosuximide; F = female; FS = febrile seizure; FS+ = febrile seizure plus; GSW = generalized spike and wave; GVG = vigabatrin; ID = intellectual disability;
IPS = intermittent photic stimulation; IQ = intellectual quotient; M = male; NA = unavailable; PA = paroxysmal activity; PB = phenobarbital; PHT = phenytoin; PIQ = performance intellectual quotient; TCS = tonic-clonic seizure(s); TIQ = full-scale
intellectual quotient; WIPPSI = Wechsler preschool and primary scale of intelligence; VIQ = verbal intellectual quotient; VPA = sodium valproate.
BRAIN 2018: 141; 3160–3178
| 3167
Motor and cognitive development were normal in six Family T included three siblings and their father, all ex-
patients (31.5%; Patients 3, 6–8, 14 and 19) with only hibiting seizures and carrying the Thr171Arg variant. All
mild language developmental delay in two, whereas 13 pro- individuals manifested febrile and afebrile seizures begin-
bands (68.4%) had a variable degree of intellectual disabil- ning in infancy and childhood with an overall benign out-
ity ranging from mild in four patients (21%; Patients 3, 4, come. One of the twin girls is still ON valproic acid (VPA)
10 and 15), to moderate in three (15.7%; Patients 5, 9 and at age 21 years and has yearly seizures; the remaining two
16) and severe intellectual disability in the remaining six siblings and the father have been seizure-free without
(31.5%; Patients 1, 11–13, 17 and 18), with autistic traits taking AEDs for several years. The brother and the father
in two. had borderline cognitive function, whereas the twin sisters
showed mild intellectual disability.
Family M1 included five family members with epilepsy, all
Families with inherited, likely carrying the Cys329Ser variant. The proband and four add-
itional symptomatic relatives spread over three generations,
pathogenic HCN1 variants exhibited infantile-onset febrile and afebrile tonic-clonic seiz-
We identified HCN1 variants segregating with epilepsy in ures. One individual manifested also a non-progressive action
four families (Table 2 and Fig. 3A), for a total of 20 indi- myoclonus; all had normal or borderline cognitive and motor
viduals (seven males and 13 females), 14 of whom ex- functions. Their EEG recordings and MRI were unremarkable.
hibited an epilepsy phenotype, while six were not known Family M2 included six family members carrying the
to have had seizures. Val414Met variant. The proband, her sister and their
HCN1 mutation spectrum BRAIN 2018: 141; 3160–3178 | 3169
maternal uncle presented late infantile, early childhood Finally, three sporadic patients (Patients 39–41) with
onset febrile and febrile tonic-clonic seizures. The remain- mild GGE and GEFS+ phenotypes, carried HCN1 missense
ing HCN1 variant carriers were not known to have had variants inherited from an asymptomatic parent. Proband
seizures. All six family members had normal development 39 had the Glu85Ala variant, inherited from his asymptom-
and those with seizures had unremarkable MRI and EEG atic mother, and exhibited GGE with tonic-clonic seizures
recordings. Only the proband is still on AEDs. at age 11 years and generalized spike wave discharges
Finally, Family O included five individuals carrying the enhanced during intermittent photic stimulation. He also
Ser680Tyr variant although only two were clinically af- manifested cortical tremor with giant evoked potentials
fected. The proband, a female with normal development, and positive C-reflex. Similarly, a 15-year-old female
because Patient 10 was an infant whereas Patient 11 was an between wild-type and homomeric channels, shifted to the
adolescent. Yet, both patients had fever-sensitive seizures and depolarizing direction ~12 mV compared to wild-type channel.
similar postnatal microcephaly (Supplementary Fig. 2). These results largely confirmed that both de novo and inherited
HCN1 variants associated with epilepsy may indifferently lead
to loss- or gain-of-function of homotetrameric HCN1 channel
Functional impact of inherited, likely properties in heterologous cells, without any immediately ob-
pathogenic variants vious correlation between phenotypes and the effects on homo-
To determine the functional impact of the variant on the meric channels.
biophysical properties of the channel, we performed whole- To gain further insight into the mechanisms by which
Figure 4 Functional impact of selected de novo HCN1 variants using whole-cell patch-clamp. (A) Representative traces of whole-
cell currents recorded in CHO cells transfected with constructs for wild-type (WT), M153I, M243R, M305L, G391D, S399P or R590Q human
(continued)
HCN1 mutation spectrum BRAIN 2018: 141; 3160–3178 | 3173
Figure 4 Continued
HCN1 channels. Currents were elicited by test pulses ranging from 20 mV to 130 mV in 10 mV increments from a holding potential of
20 mV. (B) Plot of mean current density as a function of test voltage for wild-type, M153I, M243R, K261E, M305L, G391D, S399P and R590Q
human HCN1 channels (two-way ANOVA, *P 5 0.05). (C) Mean tail current activation curves for wild-type, M153I, M243R, and R590Q HCN1
channels. (D) M153I mutant channels have faster activation time constants than wild-type HCN1 channel (two-way ANOVA, *P 5 0.05). The
M153I mutant channels also display significantly increased deactivation time constants compared with those of wild-type HCN1 (one-way
ANOVA, **P 5 0.01 for voltage 60 mV). (E) Plot of the per cent of cells expressing the Ih current for all variants. (F) Mean tail current activation
curves for wild-type, M153I and wild-type/M153I HCN1 channels. (G) Plot of the per cent of cells expressing the Ih current for all variants tested.
Data are presented as means SEM with the numbers of experiments for each condition indicated in parentheses. Values for half activation
potential (V1/2) and inverse slope factor (k) are reported in Supplementary Table 4.
3174 | BRAIN 2018: 141; 3160–3178 C. Marini et al.
Figure 5 Functional impact of variants altering glycine 391, isoleucine 397 and methionine 305 on HCN1 homotetramers and
heterotetramers. Representative current traces recorded by whole-cell patch-clamp using the indicated voltage step protocol from HEK293
cells transiently transfected with 1 mg of plasmid DNA of HCN1 wild-type, M305L (A), G391S (D), G391C (G), G391D (J), and I397L
(M) channels, as indicated. Mean steady state current/voltage relationship (I/V) from cells transfected with wild-type channel (filled black circles);
wild-type/M305L and M305L (open and filled violet circles) (B), wild-type/G391S and G391S (open and filled blue circles) (E), wild-type/G391C
and G391C (open and filled red circles) (H), wild-type/G391D and G391D (open and filled orange circles) (K) or wild-type/I397L and I397L
(continued)
HCN1 mutation spectrum BRAIN 2018: 141; 3160–3178 | 3175
more prompt to firing. We speculate that this effect in the have stronger impact on channel function whereas variants
thalamocortical network similarly results in abnormal gen- segregating with milder phenotypes were located in extra-
eralized spike-wave activity, a hallmark of GGE, observed cellular loops or in the intracellular N- and C-terminal
in patients with HCN1 variants. HCN1 is also moderately parts of the channel and had milder effects on Ih current.
expressed in the heart (source: GTEX). Yet, contrary to Additionally, noticeable phenotypic concordance was
patients with HCN4 pathogenic variants (DiFrancesco, observed in pairs of patients harbouring identical de novo
2013), no obvious cardiac abnormalities have been yet re- HCN1 variants, indicating that the epilepsy phenotype is
ported in patients with HCN1 variants. This is an import- largely determined by this single variant. In particular,
ant aspect that should also be accounted for in further G391D observed in two unrelated patients, one of Italian
studies. origin and the other Portuguese, caused an almost identical
catastrophic neonatal phenotype. Both probands presented
Genotype-phenotype correlations with severe, intractable epileptic encephalopathy with daily
and impact of HCN1 pathogenic seizures from the first days of life, and never acquired psy-
chomotor skills. Seizure semiology was also similar with
variants asymmetrical tonic posturing and predominant dysauto-
Overall, HCN1 variants causing severe phenotypes tended nomic signs including apnoea and cyanosis. Two patients
to cluster within or close to transmembrane domains and with a different change (G391S) altering the same amino
Figure 5 Continued
(open and filled green circles) (N). Mean activation curves of wild-type, M305L (C), G391S (F), G391C (I) and I397L (O) channels, both in homo-
and heterotetrameric conditions. Lines show data fit to a Boltzmann function yielding half activation potential (V1/2) and inverse slope factor
(k) values reported in Supplementary Table 4. (L) Histogram showing the percentage of cells expressing current of wild-type (100%), G391D (0%)
and wild-type/G391D (25%). The percentage of expression for all other variants are shown in Supplementary Fig. 5. All values are reported as
mean SEM.
3176 | BRAIN 2018: 141; 3160–3178 C. Marini et al.
acid both showed milder phenotypes consistent with disorder with HCN1 variants could be related to the inter-
GEFS+ spectrum. The three different changes altering gly- action of HCN1 channels with SHANK3, a scaffolding
cine 391 show that the phenotype is not only determined protein highly enriched in the post-synaptic density of glu-
by the position of the altered amino acid on the channel tamatergic synapses, whose genetic alterations have been
but also by the nature and consequence of the amino acid linked to autism spectrum disorder (Yi et al., 2016; Zhu
change. The impact of G391S/C, associated with milder et al., 2018). Finally, HCN1 missense variants located in
phenotypes, was limited to shifts in the voltage dependency parts of the channel more tolerant to variations (e.g. N-/C-
of the channel, whereas G391D, causing the most severe terminal domains) seem associated with a lower penetrance
phenotype, corrupted channel function in a more complex of epilepsy/intellectual disability phenotypes. Inherited
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